Process for the manufacture of ajoene derivatives

ABSTRACT

The present invention relates to the compound (E,Z)-ajoene of formula (1) for use in treatment of bacterial infections. Another aspect of the present invention is a composition comprising (E,Z)-ajoene of formula (1) and at least one antibiotic. Yet another aspect of the invention relates to a method for manufacturing (E,Z) ajoene of formula (1) wherein the conformation of the internal C═C— bond can be either E or Z or a mixture thereof, said method comprising reacting allicin of formula (3) with an acid in the presence of a solvent to provide (E,Z ajoene) of formula (1) as defined above. Yet another aspect of the invention is (E,Z)-ajoene of formula 1 obtainable by the method described above.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a chemical process the end product ofwhich exhibits direct medical applications. The product of the chemicalprocess known as ajoene has been found highly useful for the treatmentof bacterial infections comprising biofilm forming bacteria by blockingexpression of important quorum sensing controlled virulence factors. Theinvention also relates to a product obtainable by the above chemicalprocess which shows synergistic antimicrobial effects with antibiotics.In particular the present invention relates to the synthesis of ajoenederivatives via an oxidation of diallyldisulphide to allicin which issubsequently treated with an acid to obtain ajoene E,Z isomers in agiven ratio. Ajoene may be subjected to oxidation to obtain oxidizedvariations. Further aspects of the invention involve a composition ofajoene derivatives with an antibiotic and the use of ajoene derivativesfor the treatment of bacterial infections comprising biofilm formingbacteria.

BACKGROUND OF THE INVENTION

Bacterial infections are an increasing problem worldwide. The widespreaduse of conventional antibiotics has provoked the development ofincreasingly resistant bacterial strains along with a depleting arsenalof sufficiently effective antibiotics.

As a result, research towards a deeper understanding of the mechanismsinvolved in bacterial infections is increasing. Thus, it has beendiscovered that a wide variety of bacteria coordinate their behaviourthrough cell-to-cell communication mediated by small, diffusiblesignals. This phenomenon has been dubbed quorum sensing (QS) [W. C.Fuqua et al., J. Bacteriol., 1994, 176, p 269-275], and is prevalentamong bacteria that form complex surface attached communities calledbiofilms. It is estimated by the National Health Institute of Americathat 80% of persistent bacterial infections involve biofilms. QS enablesbacteria to keep track of their numbers, and is considered to affordthem a mechanism for minimizing host response by delaying the productionof virulence factors until sufficient bacteria have been amassed tooverwhelm host-defence mechanisms. Blocking of QS (either completely orpartly) reinstates proper action of the host-defence system whichsubsequently eliminates bacterial intruders. Therefore, inhibition of QSpresents an alternative therapeutic approach to the traditionalantibiotic-mediated bacterial killing or growth inhibition.

It has been shown that garlic extract blocks QS and promotes rapidclearing of pulmonary infections caused by the bacterial strainPseudomonas aeruginosa [T. Bjarnsholt et al., Microbiology, 2005, 151,p. 3873-3880]. Pseudomonas aeruginosa forms biofilm communities andutilizes QS through the production of signal molecules such as N-acylhomoserine lactones (AHL) and quinolones PQS). Garlic extract is also awell known natural medicine product having antibacterial and cholesterollowering effects, among others.

Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide] has previouslybeen shown to be an active ingredient in the antimicrobial garlicextracts along with allicin and other organosulfur derivatives. However,in a study on ajoene (the E:Z=1:4 isomer used), although a strong growthinhibitor of Gram positive bacteria, ajoene was shown to have a variedinhibitory effect on Gram negative bacteria, and no measurable growthinhibitory effect on P. aeruginosa [R. Naganawa et al., Applied andEnvironmental Microbiology, 62, 1996, p. 4238-4242].

Present processes for the manufacture of ajoene derivatives include theisolation of ajoene derivatives or precursors from garlic extract andvarious synthetic approaches. Isolation from garlic is highly tediousand inefficient as the amount of ajoene available pr. Kg raw garlic isminute i.e. in the range of 10 mg and HPLC separation from relatedcompounds in garlic is a necessity. Some synthetic and semi-syntheticapproaches to ajoene and various derivatives have been disclosed in theprior art. Eric Block and co-workers have disclosed a synthetic approachstarting from an oxidation of diallyl disulphide or derivatives andsubsequent heating of the resulting crude allicin or derivatives to formajoene and derivatives [EP 185324 and Block et al., J. Am. Chem. Soc.,1986, 108, p. 7045-7055]. Apitz-Castro and co-workers have presented asemi-synthetic approach where allicin (allyl 2-propenethiosulfinate) isisolated from garlic bulbs and subsequently heated and treated with alower alkyl alcohol to produce (E,Z)-ajoene derivatives [U.S. Pat. No.4,665,088]. Recently a method of converting extracted allicin to ajoeneusing acetic acid in acetone was also reported, however crude impurestarting material was used (i.e. crude allicin extracted from garlic)[WO 2010/100486]. However, the yields in the above methods have oftenbeen poor or purification by HPLC chromatography has been necessaryleading to low yields of ajoene at high purification costs. Furthermorethese synthetic approaches often lack control of the (E,Z)-isomericoutcome of the reaction, while still maintaining high yields.

Consequently, an alternative and improved method for the manufacture ofajoene derivatives would be advantageous.

It has now surprisingly been found that the synthesis of ajoenederivatives can be achieved in higher yields and better crude puritiesthan previously obtained, while being able to vary the ratio of (E,Z)isomers in the final product via specific variations in the reactionconditions. Also, it was surprisingly found that certain mixtures ofajoene derivatives and/or isomers including those obtained by the abovesynthetic process and applying a single chromatographic purificationstep were effective QS inhibitors of biofilm forming bacteria, both whenapplied alone and in composition with conventional antibiotics.

SUMMARY OF THE INVENTION

Thus, an object of the present invention relates to providing asynthetic procedure for the effective production of ajoene derivativesor specific mixtures thereof which are applicable in the treatment ofbacterial infections using QS inhibition.

In particular, it is an object of the present invention to provide aprocess for the manufacture of (E,Z)-ajoene[(E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide] in controllable E:Zratios that solves the above mentioned problems of the prior art withlow product yields and time consuming and expensive purificationprocedures.

Thus, one aspect of the present invention is (E,Z)-ajoene of formula (1)for use in treatment of bacterial infections.

Another aspect of the present invention is a composition comprising(E,Z)-ajoene of formula (1) and at least one antibiotic.

Yet another aspect of the invention relates to a method formanufacturing (E,Z) ajoene of formula (1)

wherein the conformation of the internal —C═C— bond can be either E or Zor a mixture thereof, said method comprising reacting allicin of formula(3)

with an acid in the presence of a solvent to provide (E,Z ajoene) offormula (1) as defined above.

Yet another aspect of the invention is (E,Z)-ajoene of formula (1)obtainable by the method described above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an exemplary scheme depicting the synthetic route towards(E,Z)-ajoene derivatives, including control of E:Z ratio of isomers. Thereaction conditions conditions were i) Dimethyldioxirane (DMDO),acetone, −50° C. to −20° C., 30 min, 96% yield; ii) either A) 20% AcOH,40% aqueous acetone, 64° C., 4 h, 36% yield, (Z:E −4:1) or B) 20% AcOH,40% toluene, 40° C., 48 h, 32% yield, (Z:E −1:10).

FIG. 2 shows an exemplary scheme depicting the synthetic route towardsfurther oxidized derivatives of ajoene. The reaction conditions wereiii) 4 equivalents of peracetic acid in dichloromethane at 0° C. to roomtemperature for 12 h, 38% yield or 4 equivalents of DMDO in acetone at−10° C. for 6 h at 82% yield. iv) Potassium permanganate in acetone at−20° C. for 2 h at 96% yield. v) 2 equivalents of DMDO in acetone at−10° C. for 6 h giving 78% yield. vi) Lithiated sulphide (PhSLi) in THFat −78° C. to 0° C. for 1 h giving 86% yield.

FIG. 3 shows a comparison of fold change (− indicates down regulation)in gene expression of rhIA and lasB as measured by RT-PCR (dark greybars) and DNA microarray (light grey bars). Data represent the averageof tree individual experiments. A star (*) indicates P<0.05, Student'st-test. Error bars are mean±SD.

FIG. 4 shows total rhamnolipid concentration in untreated (“no add”) andajoene treated planktonic grown P. aeruginosa. The cultures were grownin medium supplemented with 10 μg/ml, 20 μg/ml, 40 μg/ml and 80 μg/ml ofajoene (at 40 μg/ml and at 80 μg/ml the rhamnolipid content is below thedetection level at OD=1.5). Samples retrieved at OD600=1.5 (dark greybars), and at OD600=2.0 (light grey bars). Data represent the average oftree individual experiments. Error bars are mean±SD.

FIG. 5 Shows combined fluorescence and light microscopic investigationsof biofilms of P. aeruginosa exposed to PMNs (a single PMN pointed outby the arrow) at day four for 180 min at 37° C. and then subsequentlystained with the DNA stain propidium iodide (PI). A) The biofilm hasgrown without ajoene in the medium. B) The biofilm has grown in thepresence of 100 μg/mL ajoene in the medium. Red fluorescence indicatesnecrotic PMN's leaking out their content of DNA (as stained by PI).Green fluorescence indicates top areas of the P. aeruginosa biofilm.

FIG. 6 shows biofilms of P. aeruginosa PAO1 (light grey) (A) and aclinical P. aeruginosa isolate CF438 (light grey, stained with syto9)(B) at day four after 24 hours with 10 μg/ml tobramycin treatment. Deadcells are stained with the DNA stain PI (Dark grey/black). The biofilmwere visualized with CSLM. C) Implant model treated with ajoene andtobramycin.

FIG. 7 shows combined results of three separate experiments of ajoenetreatment versus no treatment (placebo) using the pulmonary infectiousmouse model. Mice were sacrificed on day one or day three post-infectionand the contents of bacteria in the lungs were determined. Open squaresindicate bacterial lung content per lung for each individual mouse. Themedian values are indicated with a filled black square. The statisticsignificance of difference in clearance was tested by a Mann-Whitney Utest (analysis of nonparametric data) and p-values for the difference atday one and day three were 0.9 and 0.002 respectively.

FIG. 8 shows results of experiments of treatment with HPLC purifiedajoene (top graph, ajoene purity >99.5%) and ajoene which have not beenHPLC purified (bottom graph, purity 95-99.5%) versus no treatment(placebo) using the pulmonary infectious mouse model. Mice weresacrificed on day three post-infection and the contents of bacteria inthe lungs were determined.

The present invention will now be described in more detail in thefollowing.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Prior to discussing the present invention in further details, thefollowing terms and conventions will first be defined:

In the context of the present invention the term (E,Z)-ajoenederivatives or (E, Z)-isomers of ajoene derivatives refers toderivatives of ajoene which are either isolated as substantially pureE-ajoene derivatives, substantially pure Z-ajoene derivatives or asmixtures of E- and Z-ajoene derivatives. The (E,Z) designations refersto the internal —C═C— double bond of ajoene as shown in the structuresby a wavy line where the configuration may vary. The term “derivatives”refers to both the two isomers (E) and (Z) and mixtures thereof and alsoto oxidized and/or reduced derivatives of (E,Z) ajoene and mixturesthereof.

An oxidizing agent, as defined herein, is any chemical reactant capableof changing the oxidation state of a molecule from its originaloxidation state to a higher oxidation state. Similarly a reducing agentis any chemical reactant capable of changing the oxidation state of amolecule from its original oxidation state to a lower oxidation state.

In the context of the present invention an acid comprises both inorganicand organic acids. This includes organic acid anhydrides or any otherpro-acids. As defined herein a pro-acid is any compound capable ofconverting into an acid under certain conditions. Such conditions couldinclude heating or subjection to an aqueous environment. Any mixture ofacids is also inferred.

As described herein biofilm refers to any aggregated, often but notnecessarily a surface bound (sessile) community of bacteria embedded ina biopolymeric matrix showing increased resistance e.g. to antibioticsand important parts of the innate immune system, as compared to theirplanktonic counterparts.

In the context of the present invention the term quorum sensing (QS)refers to a bacterial behavioural coordination mechanism operating bymeans of cell-to-cell communication transmitted by low molecular weight,diffusible signal molecules. QS systems play an important role duringthe initial event of an infection. By employing three QS systems(denoted Las, Rhl and PQS) to control expression of its impressivearsenal of virulence factors many of which are antigenic determinants,certain bacteria such as P. aeruginosa is able to operate in a stealthymanner until a certain cell density is reached where the QS system isactivated and virulence and immune protection is switched on. Uponactivation of the QS system, a coordinated release of tissue damagingand immune defence degrading virulence factors takes place.

Also in the present context N-acyl-homoserine lactone producing bacteriais defined as bacteria utilising N-acyl-homoserine lactones as messengermolecules in their cell-to-cell communication during quorum sensing.

As defined herein a quorum sensing inhibitor or QS-inhibitor is asubstance or mixture of substances capable of inhibiting bacterial QSbased communication. Blockage of QS processes either by mutation inregulatory genes or by a QS inhibitor makes said bacteria morevulnerable to both conventional antibiotics, in particularaminoglycosides such as tobramycin and important components of thecellular host-defence such as PMN-leukocytes(PolyMorphoNuclear-leukocytes).

As mentioned above the first aspect of the present invention provides amethod for making compounds of general formula (1)

also referred to as (E,Z)-ajoene. The conformation of the internal —C═C—bond can be either E or Z or a mixture thereof. The method comprisesproviding an intermediate compound of formula (3) also referred to asallicin

and treating said compound of formula (3) with an acid in the presenceof a solvent to provide compounds of formula (1) as defined above. In apreferred embodiment the concentration of the solution containingcompound (3) may be 0.06-6.0 M, preferably 0.12-3.0 M, 0.24-1.5 M,0.48-0.8 M, such as 0.6 M. The acid can be added in an excess orstoichiometric amount, but may typically be added in a catalytic amountsuch as 5-50 mol %, 10-40 mol %, 15-30 mol %, such as 20 mol %. In apreferred embodiment the acid added in the conversion of intermediate(3) to compounds of formula (1) may be a a number of acids, such ascarboxylic acids including acetic acid, propionic acid, butyric acid,pentanoic acid, hexanoic acid, or trifluoroacetic acid (TFA), orp-toluenesulfonic acid (TsOH), methanesulfonic acid (MsOH),camphorsulphonic acid (CSA). The preferred acid is acetic acid. Thechoice of acid has impact on the obtained ratio of E:Z isomers, i.e. theacid used and the corresponding E:Z ratio obtained may be for exampletrifluoroacetic acid (E:Z=about 1:1), p-toluenesulfonic acid (E:Z=about1:4), methanesulfonic acid (E:Z=about 1:4), and camphorsulphonic acid(E:Z=about 1:5), when using 40 V/V % aqueous acetone as the solvent.

In a preferred embodiment of the acid treatment providing the compoundof formula (1), the solvent used is 10-70 V/V % aqueous acetone,preferably 20-60 V/V %, 30-50 V/V %, such as 40 V/V % aqueous acetone(i.e. 60% acetone in 40% water). After addition of the acid the reactiontemperature may be adjusted to be in the range of 0-100° C., preferably20-80° C., such as 40-70° C., preferably 50-65° C., such as 64° C. Thereaction time can be from 1-24 h, preferably 2-10 h, such as 3-5 h,preferably 4 h. The use of aqueous acetone as solvent provides thecompounds of formula (1) in an E:Z ratio of 1:2-1:6, preferably 1:3-1:5,such as about 1:4, when using acetic acid in the acid treatment.

In yet another preferred embodiment of the methods described aboveproviding the compound of formula (1), the solvent used during the acidtreatment of intermediate (3) is toluene or aqueous benzene. The aqueousbenzene can be 1-50 V/V % aqueous benzene, preferably 5-30 V/V %, suchas 7-20 V/V %, preferably 10 V/V % aqueous benzene (i.e. 90% benzene in10% water). After addition of the acid the reaction temperature may beadjusted to be in the range of 0-100° C., preferably 10-80° C., such as20-60° C., preferably 30-50° C., such as 40° C. The reaction time forthe acid treatment in toluene or aqueous benzene can be from 2-96 h,preferably 24-72 h, such as 36-60 h, preferably 48 h. The use of tolueneor aqueous benzene as solvent provides the compounds of formula (1) inan E:Z ratio of 6:1-20:1, such as about 10:1, when using acetic acid inthe acid treatment.

The purification of the obtained products may typically be performedusing standard solvent extraction techniques followed by columnchromatographic purification, but other methods may also be used such asdistillation, preparative thin layer chromatography or crystallizationfor solid compounds.

In a preferred embodiment of the purification of compound (1) thereaction mixture is cooled to room temperature and diluted with 3volumes of a 1:1 mixture of water and methanol followed by an extractionwith an apolar solvent such as pentane. The aqueous fraction may besaturated with ammonium sulphate and further extracted with a more polarsolvent than pentane such as dichloromethane. (E,Z)-ajoene (1) can beisolated by concentrating the combined organic extracts and subjectingthe crude product to silica column chromatography using an eluent mobilephase comprising 40-99 V/V % ethyl acetate in pentane, preferably 50-90V/V %, 60-80 V/V %, such as 70 V/V %.

The purity of (E,Z)-ajoene after the above procedure may be 95-99.5%,such as 97-99.5%, 98-99.5%, 99-99.5% such as about 99.5%. It wassurprisingly found by the inventors, that upon further purification ofthe product obtained after column chromatography, e.g. using reversephase HPLC chromatography, or other purification steps, said(E,Z)-ajoene product lost all or parts of its biological activity, ine.g. the assays as described in the examples. This was also found whenisolating pure (E)-ajoene and/or pure (Z)-ajoene, e.g. using HPLC. It isspeculated that small amounts of ajoene derived chemistry, i.e.compounds related to ajoene, such as less than 5%, 3%_(,) 2%, 1%, suchas less than 0.5% may be highly important in the stabilisation of(E,Z)-ajoene and derivatives, towards degradation, e.g. oxidation,reduction and or cleavage, both in vitro but importantly also in vivo.

One embodiment of the invention is a method as described above whereinthe (E,Z)-ajoene of formula (1) is subjected to no more than onechromatographic purification step, said chromatographic purificationstep not comprising reversed phase HPLC.

After purification the (E,Z)-ajoene derivatives can be isolated asmixtures of isomers in yields based on compound (3) of at least 10 mol%, preferably 15 mol %, 20 mol %, 25 mol %, such as at least 30 mol %.

In one embodiment the allicin of formula (3) may be manufactured bytreating a compound of formula (2), also referred to asdiallyldisulphide (DADS)

with an oxidizing agent. In a useful embodiment of the method, theoxidizing agent used to oxidize compounds of formula (2) to compounds offormula (3) may be selected from the group consisting of DMDO, MCPBA,peracetic acid, potassium permanganate, magnesium oxide and Swernoxidants (e.g. oxalyl chloride, dimethyl sulfoxide (DMSO) and an organicbase, such as triethylamine) DMDO is a preferred oxidant as it providesthe highest yields. The oxidizing agent may be added in catalytic orstoichiometric amount relative to compound (2) but preferably in anexcess amount such as 1.01-10.0 mol equivalents, preferably 1.05-5.0 molequivalents, such as 1.07-2.0 mol equivalents, preferably 1.10 molequivalents. The compound of formula (2) may be advantageously added toa solution of the oxidizing agent at low temperature over a period ofseveral minutes. The concentration of oxidizing agent in the solutioncan be 0.001-1.0 M, preferably 0.005-0.5 M, 0.01-0.1 M, such as 0.07 M.The solution may be in a solvent capable of staying fluid at lowtemperatures, such as acetone, dichloromethane, chloroform,tetrachloromethane, diethylether, ethyl acetate, or tetrahydrofurane.The solution temperature while adding compound (2) may be −100° C. to10° C., preferably −80° C. to −10° C., −60° C. to −40° C., such as −50°C. Compound (2) may be added over a period of 1-120 min, preferably 5-90min, 7-60 min, 10-30 min, 12-20 min, such as 15 min. After addition ofcompound (2) to the solution, this may advantageously be allowed to warmup slowly by 10-60° C., preferably 20-40° C., such as 30° C., over aperiod of 10-120 min, preferably 15-90 min, 20-60 min, 25-40 min, suchas 30 min. Concentration via evaporation of solvents provides theintermediate compound (3) in a crude form with a purity of at least 80mol %, preferably at least 85 mol %, at least 90 mol %, such as at least95 mol %.

The crude allicin (3) may be used without further purification, andstill provide good yields in the following step. This could be due tothe lack of acid present in the oxidation procedure, which seems tocontribute to degradation of allicin during oxidation and subsequentstorage. However, in a preferred embodiment allicin (3) mayadvantageously be purified in order to isolate a substantially allicin(3) prior to the subsequent acid treatment. This was found to provide aneven cleaner reaction and higher yields in the subsequent acid treatmentstep to provide compounds of formula (1). The purification can beperformed using any conventional purification methods, preferably silicacolumn chromatography. Chromatography can be performed using a mobilephase eluent consisting of a mixture of diethyl ether in pentane,preferably 0.5-20 V/V % diethylelether in pentane, such as 1-10 V/V %,2-8 V/V %, 3-7 V/V %, 4-6 V/V %, preferably 5 V/V %. Thereby a compoundof formula (3) is obtained in a yield based on compound (2) of at least80 mol %, preferably at least 85 mol %, at least 90 mol %, such as atleast 95 mol %.

In a second aspect of the invention a process for making compounds offormula (4) is provided

where Y is chosen from —S—, —S(O), or —S(O₂)—, R is chosen from allyl,phenyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, benzyl, p-tolouyl,m-tolouyl, o-tolouyl, and the conformation of the internal —C═C— bondcan be either E or Z or a mixture thereof. This process comprises themethod as defined above to make compound of formula (1) followed by anadditional reaction step involving reacting (E,Z) ajoene of formula (1)with at least one additional oxidizing reagent to provide a compound offormula (4) wherein R is allyl, and optionally reacting the compound offormula (4) with an R substituted lithiated sulphide (R-SLi) to providecompounds of formula (4) wherein R is phenyl, methyl, ethyl, propyl,butyl, pentyl, hexyl, benzyl, p-tolouyl, m-tolouyl, or o-tolouyl.

In one embodiment of the method defined above, the second oxidizingagent used to oxidize compounds of formula (1) to compounds of formula(4) may be selected from the group consisting of DMDO, MCPBA, peraceticacid, potassium permanganate, Hydrogen peroxide, Bis TMS Peroxide, orany mild peroxides. The reaction with R-substituted lithiated sulphidemay be performed in tetrahydrofurane solvent at −78° C. up to 0° C.Reaction time may be 30 min to 24 hours, preferably 30 min to 2 hours.

The purification of compounds of formula (4) may be performed in asimilar manner to that of compounds of formula (1) as described above.

The compounds obtained of general formula (4) can be isolated asmixtures of (E,Z)-isomers, or these isomers can be separated to providesubstantially pure (E)-isomers or substantially pure (Z)-isomers. Also,the compounds of formula (4) having different oxidation states of Y,i.e. Y equal to —S—, S(O), or —S(O₂)— can be either separated orisolated as mixtures.

Another embodiment of the present invention is the compounds of formula(1) obtained by the methods of described above or alternatively thecompounds of formula (1) obtainable by the methods of described above.As described, the above method leads to good crude purities, which leadsto the use of only one chromatographic purification step, which hasproved to be vital to obtain a more biologically active (E,Z)-ajoenederivative (1). Such active ajoene derivatives may be said to have apurity in the range of 95-99.5%, such as about 97-99.5%, 98-99.5%,99-99.5% such as about 99.5%. The E:Z ratio of the (E,Z) ajoene obtainedmay preferably be from 20:1 to 1:10, such as from 15:1 to 1:6, 12:1 to1:5, preferably from about 10:1 to about 1:4. Alternatively the E:Zratio may preferably be from 1:1 to 1:10, such as from 1:2 to 1:8, 1:3to 1:5, preferably about 1:4.

Yet a further aspect of the invention is a composition comprising a(E,Z)-ajoene having formula (1) and at least one antibiotic. Thecompound (i.e. (E,Z)-ajoene having an obtainable E:Z ratio includingpure E or pure Z) may be obtained by the method described above, or anyother methods providing a biologically active QS-inhibiting (E,Z)-ajoeneproduct. Such compositions may be used in the formulation of medicamentsand thus may be formulated as a medicament or dosage form comprising a(E,Z)-ajoene having formula (1), at least one antibiotic andpharmaceutically acceptable carriers and/or binders. The medicament maybe formulated as liquid or solid dosage forms, where liquid compositionsmay include liquid compositions for topical administration, liquidcompositions for intravenous injection, intramuscular or subcutaneousinjection, or a liquid composition for inhalation as an aerosol. Soliddosage forms may include tablets, capsules, powders, including powdersfor inhalation. Embodiments wherein the (E,Z)-ajoene is administered inone of the above ways, while the antibiotic is administered in any otherof the above ways are also inferred. For example the ajoene may beadministered by inhalation, via powder or aerosol, while the antibioticis administered intravenously.

The E:Z ratio of the (E,Z) ajoene in the composition may preferably befrom 20:1 to 1:10, such as from 15:1 to 1:6, 12:1 to 1:5, preferablyfrom about 10:1 to about 1:4. Alternatively the E:Z ratio may preferablybe from 1:1 to 1:10, such as from 1:2 to 1:8, 1:3 to 1:5, preferablyabout 1:4.

One embodiment is thus a composition as described above comprising(E,Z)-ajoene having formula (1) and at least one antibiotic for use inthe treatment of infections, preferably bacterial infections comprisingbiofilm forming bacteria.

An advantage of such compositions is that a synergistic effect isachieved when using a QS-inhibitor in a composition with an antibiotic,as demonstrated herein. In the context of the present invention anantibiotic is defined as any antibacterial substance where theantibacterial effect is not based on QS inhibition. Typically, theantibiotic used in combination with a QS-inhibitor can be classified aseither bactericidal or bacteriostatic. Bactericidals kill bacteriadirectly where bacteriostatics prevent bacteria from dividing. Classesof antibiotics include aminoglycosides, ansamycins, carbacephem,carbapenems, cephalosporins, glycopeptides, macrolides, monobactams,penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines.The antibiotic may be selected from any of these classes.

The antibiotic may be selected from the group consisting of Ampicillin,Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin,Ticarcillin, Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam,Benzylpenicillin, Cloxacillin, Dicloxacillin, Methicillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin Tazobactam, TicarcillinClavulanic Acid, Nafcillin, Cefadroxil, Cefazolin, Cephalexin,Cephalothin, Cephapirin, Cephradine, Cefaclor, Cefamandol, Cefonicid,Cefotetan, Cefoxitin, Cefprozil, Ceftmetazole, Cefuroxime, Loracarbef,Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoximeproxetil, Ceftazidime, Ceftizoxime, Ceftriaxone, Cefepime, Azithromycin,Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin,Troleandomycin, Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin,Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid,Gemifloxacin, Perfloxacin, Imipenem-Cilastatin, Meropenem, Aztreonam,Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin,Tobramycin, Paromomycin, Teicoplanin, Vancomycin, Demeclocycline,Doxycycline, Methacycline, Minocycline, Oxytetracycline, Tetracycline,Chlortetracycline, Mafenide, Silver Sulfadiazine, Sulfacetamide,Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole, Sulfamethizole, Rifampin, Rifabutin,Rifampin, Rifapentine, Linezolid, Quinopristin Dalfopristin, Bacitracin,Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazol,Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin,Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin,Ethionamide, Pyrazinamide, Para-aminosalicyclic acid, and Erythromycinethylsuccinate. The antibiotic may e.g. be tobramycin.

One aspect of the present invention is (E,Z)-ajoene of formula (1) foruse in treatment of bacterial infections, preferably bacterialinfections comprising biofilm forming bacteria.

Another aspect of the invention is (E,Z)-ajoene of formula (1) asobtained by the herein described method for use in treatment ofbacterial infections, preferably bacterial infections comprising biofilmforming bacteria.

The below described embodiments thus relates to both (E,Z)-ajoene ingeneral, and also to (E,Z)-ajoene as provided in the herein describedmethod and finally also to compositions comprising (E,Z)-ajoene and anantibiotic as described above.

The E:Z ratio of the (E,Z) ajoene obtained may preferably be from 20:1to 1:10, such as from 15:1 to 1:6, 12:1 to 1:5, preferably from about10:1 to about 1:4. Alternatively the E:Z ratio may preferably be from1:1 to 1:10, such as from 1:2 to 1:8, 1:3 to 1:5, preferably about 1:4.

The treatment of bacterial infections, including those comprisingbiofilm forming bacteria, may be prophylactic. The compounds used may bein a composition with an antibiotic and/or a pharmaceutically acceptablecarrier. The compounds or compositions may be administered byintravenous injections, intramuscular or subcutaneous injections,orally, topically or they may be inhaled, preferably using a suitableinhalation device. They may be inhaled as solid compositions, such aspowders, or as liquid compositions in the form of aerosols.

In a one embodiment the bacterial infection treated is in a mammal withan immune deficiency. In the context of the present invention a mammalwith immune deficiency is a mammal that for any reason isimmuno-compromised, meaning that the performance of some or all aspectsof the natural defence is lower than normal. Typically, this may be dueto an immuno-compromising disease or due to therapy usingimmuno-suppressant medication. Such mammals are at higher risk ofinfections with biofilm forming bacteria such as Pseudomonas aeruginosaand would therefore potentially benefit from both prophylactic and acutetreatment with a QS inhibitor which may be in a composition with anantibiotic. Mammals may comprise humans, pets and livestock, or mammalsmay be selected from the group consisting of humans, pets and livestock.Immuno-compromising diseases include diseases selected from the groupconsisting of cystic fibrosis, diabetes mellitus, COPD, malignanthaematological disease, cancer, HIV, AIDS, chronic wounds, burn wounds.Patients may include patients with indwelling catheters, patientstreated with medical equipment, implants, stents and patients in ICU's.

In a preferred embodiment of the invention the bacteria, particularlythe biofilm forming bacteria causing the infections described above maybe selected from the group of bacteria capable of QS controlledvirulence such as gram negative bacteria, particularly the followinggroups of Gram negative bacteria: Vibrio fischeri, Aeromonas hydrophila,Aeromonas salmonicida, Agrobacterium tumefaciens, Burkholderia cepacia,Chromobacterium violaceum, Enterobacter agglomerans, Erwinia carotovora,Erwinia chrysanthemi, Erwinia Stewartii, Escherichia coli, Pseudomonasaureofaciens, Pseudomonas aeruginosa, Ralstonia solanacearum, Rhizobiumetli, Rhizobium leguminosarum, Rhodobacter sphaeroides, Salmonellatyphimurium, Serratia liquefaciens, Sinorhizobium meliloti, Vibrioanguillarum, Vibrio harveyi, Yersinia enterocolitica, Yersiniapseudotuberculosis, particularly Pseudomonas aeruginosa.

Yet an additional preferred embodiment of the present inventions is(E,Z)-ajoene of formula (1) for use in the treatment of bacterialinfections where the infected area is a wound, such as a chronic woundand/or non-healing wound. In these cases the compounds mayadvantageously be administered topically.

Another useful embodiment is (E,Z)-ajoene of formula (1) for use in thetreatment of bacterial infections where the infected area is the lungs.This treatment is especially relevant for cystic fibrosis patients who,due to their reduced immune response, often suffer from persistent lunginfections comprising biofilm forming bacteria, such as Pseudomonasaeruginosa. Therefore, a preferred embodiment is (E,Z)-ajoene of formula(1) for use in the treatment of bacterial infections where the infectedarea is the lungs of a cystic fibrosis patient. In these cases thecompounds may advantageously be administered via inhalation or byintravenous injections.

In another preferred embodiment the infected area is an implant or thearea around an implant. As defined herein an implant is a medical devicemade to replace or act as a missing biological structure in a mammal.Such implants may include implants containing electronics such asartificial pacemakers and cochlear implants, subcutaneous drug deliverydevices in the form of implantable pills or drug-eluting stents, aprosthetic device such as bone-replacement and support devices or dentalimplants.

In yet another embodiment the infected area may be the part of thedigestive system, such as the stomach and/or intestines. Such infectionsmay include food poisoning or infections arising from pancreatic orbiliary stents.

It should be noted that embodiments and features described in thecontext of one of the aspects of the present invention also apply to theother aspects of the invention. Also, all of the above embodiments abovedescribing compounds and compositions for use in the treatment ofvarious indications, may all equally be described as methods of treatingsaid indication by administration of the said compounds or compositions.

All patent and non-patent references cited in the present application,are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the followingnon-limiting examples.

EXAMPLES Example 1 Synthesis of Compound (3), Allicin

An acetone solution of DMDO (31 mL, 0.07M, 2.2 mmol) was added dropwiseover 15 min to compound (2), diallyldisulphide (0.29 g, 2.0 mmol) inacetone (1 mL) at −50° C. under argon. The resulting light yellowreaction mixture was allowed to stir for 30 min while slowly warming to−20° C. The resulting clear reaction mixture was concentrated in vacuo.The crude residue (0.31 g, 98%) was further purified by columnchromatography (eluting with 5% Et₂O in pentane) to yield compound (3),allicin (0.31 g, 1.9 mmol, 96% yield, purity >95% and up to 99.9%) as alight yellow liquid.

The above reaction was repeated under varying condition with respect tosolvent and temperature. Remaining parameters were kept the same. Asseen in table 1 below the reaction is effective under a number ofconditions.

TABLE 1 Conversion of diallyldisulphide to allicin (3) Entry SolventTemp 2/Yield 1 Acetone −100° C., 78° C., −50-−20° C. 96% 2 CH₂Cl₂ −100°C., 78° C., −50-−20° C. 90% 3 CHCl₃ −78-−60° C. 92% 4 CCl₄ −78-−60° C.96% 5 Et₂O −50-−20° C. 86% 6 EtOAc −50-−20° C. 80% 7 THF −50-−20° C. 85%

Example 2 Synthesis of Compound (1), (E,Z)-Ajoene Using Acetone Solvent

A solution of compound (3), allicin (0.162 g, 1.0 mmol) in a 40% aqueousacetone solution (1.6 mL) was treated with AcOH (0.011 mL, 0.2 mmol).The resulting mixture was heated to 64° C. for 4 h. The cooled reactionmixture was diluted with 50% aqueous methanol (6 mL) and extracted withpentane (5×10 mL). The aqueous fraction was saturated with solid NH4SO4and the mixture was extracted with CH₂Cl₂ (5×10 mL). The combinedorganic extracts were dried (Na₂SO₄) and concentrated in vacuo. Thecrude residue (0.16 g) was further purified by column chromatography(eluting with 60-80% EtOAc in pentane) to yield compound (1),(E,Z)-ajoene (E:Z ratio=1:4, 0.025 g, 0.1 mmol, 32% yield) as a lightyellow liquid.

The above reaction was repeated using a variety of acids. Remainingparameters were kept the same as above. Exchange of acid had impact onboth yields and E:Z ratios of ajoene as seen in table 2 below:

TABLE 2 conversion of allicin (3) to (E,Z) ajoene (1) using differentacids. Entry Acid Conditions Yield (E:Z) 1 TsOH 40% aq. acetone, 64° C.,4 h 24 (1:4) 2 CSA 40% aq. acetone, 64° C., 4 h 20 (1:5) 3 MsOH 40% aq.acetone, 64° C., 4 h 22 (1:4) 4 TFA 40% aq. acetone, 64° C., 4 h 12(1:1)

Importantly, it was found that when using less than highly pure allicin(3) the yields in the above reactions were drastically reduced, if notzero. It was also found that when using the methods of the prior art toobtain allicin (3), some impurities stemming from either garlic extractor the reactants or the products used in the block synthesis of allicinresulted in yields of ajoene no higher than 6-12%. This appliedregardless of which solvent and acid was used for the ajoene synthesis.

Example 3 Synthesis of Compound (1), (E,Z)-Ajoene [E:Z=10:1]

A solution of compound (3), allicin (0.162 g, 1.0 mmol) in a 10% aqueousbenzene solution (1.6 mL) was treated with AcOH (0.011 mL, 0.2 mmol).The resulting mixture was heated to 37° C. for 48 h. The cooled reactionmixture was diluted with 50% aqueous methanol (6 mL) and extracted withpentane (5×10 mL). The aqueous fraction was saturated with solid NH₄SO₄and the mixture was extracted with CH₂Cl₂ (5×10 mL). The combinedorganic extracts were dried (Na₂SO₄) and concentrated in vacuo. Thecrude residue (0.15 g) was further purified by column chromatography(eluting with 60-80% EtOAc in pentane) to yield compound (1)(E,Z)-ajoene (E:Z ratio 10:1, 0.023 g, 0.1 mmol, 30% yield) as a lightyellow liquid.

Example 4 Synthesis of a Compounds of Formula (4)

Oxidized sulphone derivatives of (E,Z) ajoene has been shown to have QSinhibitory effects. Therefore such derivatives, i.e. compounds offormula (4) were synthesized using various routes as described below(see also FIG. 2):

Y=S, R=allyl

The compound of formula (1) ((E,Z) Ajoene) was treated with potassiumpermanganate (KMnO₄) in acetone at −20° C. for 2 hours to obtain acompound of formular (4) with Y=S and R=allyl in 96% yield. Workup andpurification was analogous to that used for (E,Z) ajoene.

Y=S(O)₂, R=allyl

The compound of formula (1) ((E,Z) Ajoene) was treated with peraceticacid in dichloromethane at 0° C. for 12 hours to obtain a compound offormular (4) with S(O)₂, R=allyl in 38% yield. Workup and purificationwas analogous to that used for (E,Z) ajoene.

Alternatively, the compound of formula (1) ((E,Z) Ajoene) was treatedwith 4 equivalents of DMDO in acetone at −10° C. for 6 hours to obtain acompound of formula (4) with S(O)₂, R=allyl in 82% yield. Workup andpurification was analogous to that used for (E,Z) ajoene.

In yet another alternative route the compound of formula (4) with Y=Sand R=allyl as described above was treated with 2 equivalents of DMDO inacetone at −10° C. for 6 hours to obtain a compound of formular (4) withS(O)₂, R=allyl in 78% yield.

Y=S, R=phenyl

The compound of formula (4) where Y=S(O)₂, R=allyl was treated withlithiated sulphide (PhSLi) in tetrahydrofurane at a temperature of −78°C. which was allowed to rise to 0° C. over 1 hour to obtain the compoundof formula (4) having Y=S, R=phenyl in 86% molar yield.

Y=S, R=methyl, ethyl, benzyl

In analogy to the method above where Y=S, R=phenyl, the correspondingcompounds of formula (4) with R=methyl, ethyl, benzyl was produced insimilar yields using the corresponding lithiated sulphides (i.e. MeSLi,EtSLi and allylSLi). Remaining parameters were kept the same.

Example 5 P. aeruginosa Genes Regulated by Ajoene Treatment

DNA microarray analysis was used to identify genes regulated by ajoenetreatment. As a reference we have used the QS regulon previouslyidentified by Hentzer et al., [M. Hentzer et al., EMBO J., 2003, 22, p.3803-3815] and these data has been used to validate target specificityof putative quorum sensing inhibiting (QSI) compounds. Exponentialgrowing P. aeruginosa cultures were treated with the following fourconcentrations of ajoene; 10 μg/ml (42.7 μM), 20 μg/ml (85.4 μM), 40μg/ml (170.8 μM) and 80 μg/ml (341.6 μM). (E,Z) ajoene having an E:Zratio of 1:4 was used. These concentrations do not exert any growthinhibitory effects. The samples were retrieved at an OD600 nm of 2.0(optical density) because previous investigations have shown the highestactivity among the QS genes at that cell density. Gene expression of thetreated cultures was compared to an untreated control.

In general, there was only a small number of genes that were downregulated more than 5 fold by the four different concentrations ofajoene; 0 genes at 10 μg/ml ajoene, 0 genes at 20 μg/ml ajoene, 2 genesat 40 μg/ml ajoene and 11 genes at 80 μg/ml ajoene. The amount of genesup regulated more than 5 fold did not vary noticeably between thetreated cultures. According to our experimental settings, expression of10 out of the total number of 163 QS controlled P. aeruginosa genes(according to Hentzer et al., [M. Hentzer et al., EMBO J., 2003, 22, p.3803-3815]) was down regulated by the ajoene treatments. A concentrationof 80 μg/ml ajoene affects expression of 11 out of a total of 5570 P.aeruginosa genes. Ajoene is therefore highly specific for a subclass ofQS controlled genes (Table 3).

TABLE 3 Alterations in gene expression by ajoene. Genes included are >5times down regulated by 80 μg/ml ajoene treatment. The numbers are foldchange in gene expression compared to an untreated control. Datarepresent the average of tree individual experiments. Gene [Ajoene]μg/ml No Gene 10 20 40 80 Description PA0852 cbpD −2.8 −2.5 −3.9 −6.9Chitin-binding protein PA1871 lasA −2.6 −2.2 −3.0 −8.7 LasA proteaseprecursor PA2069 — −2.3 −2.4 −4.0 −5.3 Probable carbamoyl transferasePA2146 — −1.3 −1.8 −2.6 −7.3 Conserved hypothetical protein PA2300 chic−2.5 −2.1 −5.1 −24.6 Chitinase PA2570 pa1L −1.8 −2.0 −3.3 −6.3 LecAPA3478 rhlB −2.6 −2.0 −3.3 −8.7 Rhamnosyltransferase chain B PA3479 rhlA−2.2 −1.5 −2.6 −8.8 Rhamnosyltransferase chain A PA4141 — −1.0 −1.1 −1.3−5.4 Hypothetical protein PA4142 — −2.0 −2.2 −2.7 −5.1 Probablesecretion protein PA4175 prpL −3.7 −3.3 −5.3 −6.8 Pvds-regulatedendoprotease *Indicates P < 0.05, **indicates P < 0.01, Student'st-test.

Among the genes significantly down-regulated by ajoene were thefollowing QS regulated important virulence factors, LasA protease (lasA,PA1871), chitinase (chiC, PA2300), the cytotoxic galactophilic lectin(lecA, PA2570), the rhamnosyl transferase AB operon (rhIA, PA3478 andrhIB, PA3479), the PvdS-regulated endoprotease (prpL, PA4175) thatdegrades casein, elastin, lactoferrin, transferrin, and decorin, and theassociated chitin-binding protein cbpD (PA0852) which mediatesattachment to chitin-containing substrates and presumably assist inbiofilm formation. lasB was not more than 5-fold down regulated.

Example 6 Verification of the Microarray Data

To verify the microarray data, RT-PCR was performed with two of the keyQS genes; lasB and rhIA (see FIG. 3). When comparing the two methods therepression of both the lasB and rhIA gene expression follow the sametrend, with a general slightly increase in fold reduction observed withthe RT-PCR based method. The RT-PCR data shows that a concentration of80 μg/ml ajoene lowers the expression of rhIA almost 12 fold and lasBalmost 5 fold. The expression of rhIA is primarily controlled by the RhlQS system and the signal molecule BHL whereas the expression of lasB isaffected by both the Las and the Rhl QS systems and the concentration ofthe signal molecules OdDHL and BHL. According to Rasmussen et al.,[Rasmussen et al., Microbiology, 2005, 151, p. 1325-1340], the geneslisted in table 3 are (except prpL which regulation is entirely governedby the Las QS system) subject to regulation by both the Las and Rhl QSsystems.

This suggests that ajoene primarily targets the Rhl QS. (E,Z) ajoenehaving an E:Z ratio of 1:4 was used in these experiments.

Example 7 Attenuation of Rhamnolipid Production by Ajoene

To exemplify the actual efficacy of ajoene in inhibiting synthesis ofone of the major virulence factors, the concentration of rhamnolipidpresent in the cultures grown for DNA array and RT-PCR where directlyquantified by LC-MS. The production of rhamnolipids is initiated earlyin stationary phase co-ordinately regulated by the Rhl QS system encodedby the rhIA, rh/B and rh/C genes (PA3479, PA3478 and PA1131) [V. E.Wagner et al., J. Bacteriol., 2003, 185, p. 2080-2095 and R. Rahim etal., Mol. Microbiol., 2001, 40, p. 708-718]. Samples where thereforeretrieved at an OD600 nm of 1.5 and 2.0 to monitor rhamnolipidproduction before and after the synthesis was fully initiated. Theconcentration of rhamnolipid in the samples was found to be graduallydecreased with increasing concentration of ajoene. When treated with 20μg/ml ajoene at an OD600 nm of 2.0, the rhamnolipid content were reducedto approximately ⅓ compared to the untreated culture and there wasalmost no detectable rhamnolipid present in the sample when the cellswere treated with 80 μg/ml ajoene. This concentration therefore almostcompletely blocks rhamnolipid synthesis. (E,Z) ajoene having an E:Zratio of 1:4 was used.

Example 8 Ajoene Treatment Renders In Vitro Biofilms RhamnolipidDeficient and Prevents the Killing of PMNs

Biofilms exposed to PMN leukocytes produce rhamnolipids that function asa shield and protects the biofilm bacteria from phagocytosis. Theinfluence of ajoene on the lysis of PMNs was demonstrated by using an invitro continuous-culture once through flow chamber biofilm system [B. B.Christensen et al., Methods Enzymol., 1999, 310, p. 20-42]. The P.aeruginosa biofilms were grown for four days either in the presence orabsence of 100 μg/ml ajoene. When PMNs subsequently were introduced inthe flow chambers on top of the biofilms grown in the absence of ajoene,propidium iodide (PI) staining indicated necrosis of the PMNs (FIG. 5A).In contrast, when the biofilm was grown in the presence of ajoene nonecrosis of the PMNs was observed (FIG. 5B). Ajoene treatment thereforerescues the PMN leukocytes by blocking biofilm synthesis of ajoene. Ithas previously been shown that rescued PMN's can phagocytose biofilmbacteria [T. Bjarnsholt et al., Microbiology, 2005, 151, p. 3873-3880].(E,Z) ajoene having an E:Z ratio of 1:4 was used.

Example 9 In Vitro and In Vivo Conformation of Synergistic Effect ofTreatment with Ajoene and Antibiotics

One important issue in the treatment of bacterial biofilm infections isthe lowered effectiveness of administered antibiotics. An infection inthe airways of CF patients will result in high concentrations of anionicpolyelectrolytes like DNA [T. Brandt et al., Thorax, 1995, 50, p.880-882] released from lysed inflammatory cells such as PMN's andbacteria. It has been showed that anionic polyelectrolytes and inparticularly DNA binds to and reduce the activity of cationicantibiotics like tobramycin which can lead to a decrease in thebiological available tobramycin to as low as 5% of the existing quantity[P. M. Mendelman, 1985, Am Rev Respir Dis., 132, p. 761-765]. Thissuggests that by blocking the production and release of rhamnolipids andeDNA, it is possible to reduce the following inactivation of tobramycin.

In concordance with that, we have previously published that combinationtreatment with garlic extract and the antibiotic tobramycin of a threeday old biofilm (grown in vitro in the continuous-culture, once-throughflow cells) showed a synergistic killing effect [T. Bjarnsholt et al.,Microbiology, 2005, 151, p. 3873-3880]. It was however unknown what wasthe active ingredient in the garlic extract. Thus. biofilms of P.aeruginosa strain were either grown in the presence or absence of 100μg/ml ajoene. At day three the biofilms were treated with 10 μg/mltobramycin for 24 hours. A prior treatment study indicated thattreatment with 10 μg/ml, 100 μg/ml or 340 μg/ml would show no differencein the extent of killing. 10 μg/ml tobramycin was therefore chosen forthe present study. The killing efficacy was evaluated using live deadstains and confocal scanning laser microscopy (CSLM) and showed almost100% killing of the biofilm grown in the presence of ajoene [ajoene hadnot been subject to e.g. HPLC purification] and subsequently treatedwith tobramycin (FIG. 6A). This synergistic effect was also evaluatedusing the clinical CF isolate CF438 (a first isolate from a diagnosed CFchild) which possesses functional QS systems and again almost 100%killing of the biofilm were detected (FIG. 6B).

To examine the effect of a combination treatment with ajoene andtobramycin in vivo we used an foreign-body infection model [L. D.Christensen et al., Microbiology, 2007, 153, p. 2312-2320]. Siliconetube implants with a size of 4 mm (inner diameter 4 mm/outer diameter 6mm) pre-colonized with wild-type P. aeruginosa, were inserted into theperitoneal cavity of mice. Treatments were injected subcutaneously every24 hours, where ajoene and tobramycin treatment were initiated two dayspre-insertion and 24 hours post-insertion, respectively. 72 hours afterinsertion the implants were removed from the mice and the number ofcolony-forming units per implant was determined. We found that treatmentof wild-type P. aeruginosa with a combination of ajoene and tobramycinresulted in a significant faster clearing of the implants as compared toboth the placebo group and the single treatments with ajoene ortobramycin (p<0.0001, p=0.009 and p=0.01, respectively). A significantdifference in clearing was also observed between the placebo group andthe single treatment groups (p=0.008 and p<0.0001). We found nosignificant difference in clearing between the two single treatmentgroups (p=30) (FIG. 6C). (E,Z) ajoene having an E:Z ratio of 1:4 wasused.

We have shown in vitro that addition of 100 μg/ml ajoene to a biofilmfollowed by addition of 10 μg/ml tobramycin kills almost 100% of thebiofilm whereas the presence of tobramycin or ajoene only results in noor only minor killing of the biofilm bacteria. It is documented that therelease of cDNA is controlled by QS which taking into consideration andcombined with our results points to a possible attenuation of therelease of cDNA by ajoene.

Example 10 Ajoene Treatment of a Pulmonary P. aeruginosa Infection

In this study ajoene was administered prophylactic and subsequentlycontinued after infection. Injections were given subcutaneous in theperitoneum. We performed three individual in vivo treatment experimentswith ajoene in the mouse model of pulmonary infection using the P.aeruginosa PAO1. BALB/c mice were intracheally challenged (at day 0)with alginate beads containing 1.5×10⁸ CFU/ml P. aeruginosa. The twogroups of mice were either untreated (placebo) or treated with ajoene 25μg/g mouse once a day. The mice were given two days of prophylactictreatment or placebo prior to bacterial challenge. Treatment wascontinued for three days. Enumeration by plate counts showed asignificant difference between the treated group and the control groupon day three. When combining the experiments a significant difference onday three was seen (p<0.002) with a 500 fold difference in clearancebetween the groups (FIG. 7). This is concordant with the resultsobtained by Bjarnsholt et al (2005) in a similar lung infection model inwith raw garlic extract was used as treatment [T. Bjarnsholt et al.,Microbiology, 2005, 151, p. 3873-3880]. The mice tolerated well theadministered dosage of 25 μg/g body weight.

Similar experiments comparing the ability of ajoene to induce clearanceof P. aeruginosa infections as described above, when used at very highpurity (>99.5%, HPLC purified) or at purities from 95% to 99.5%, (i.e.crude ajoene or ajoene subject to a single column chromatography step)demonstrated that very high purity ajoene was not effective at clearingthe pulmonary infection while 95-99.5% purity ajoene was (FIG. 8, topand bottom graph respectively). This highlights that a synthesisproviding a sufficiently pure “crude” ajoene product is essential inorder to avoid the use of HPLC purification, as this step has adetrimental effect on biological activity. Hence, the present processproviding ajoene in a purity above 95% and below 99.5% after workup andoptionally column chromatography has a surprising advantage over theprior art. (E,Z) ajoene having an E:Z ratio of 1:4 was used.

When comparing the present studies with ajoene to earlier studies withgarlic extracts, the present study offers a convincing indication ofajoene and close derivatives being the major active components in garlicable to reduce a P. aeruginosa infection.

CONCLUSION

In conclusion we have demonstrated the use of ajoene to attenuatevirulence of P. aeruginosa in vivo by inhibiting transcription of ahandful of important QS controlled virulence genes in P. aeruginosa.From our transcriptome analysis we have suggested a possible target ofajoene being the Rhl QS system. It is worth to acknowledge that QSinhibition does not remove the P. aeruginosa capability of forming abiofilm. However, all available data indicate that a QS deficientbiofilm however is more fragile compared to the QS proficient biofilm.Since for example the matrix component DNA is missing (matrix productionis Rhl QS controlled) the biofilm is sensitive to sheer forces and canslough off dependent on the hydrodynamic forces. In addition, sincerhamnolipid is not formed, the biofilm becomes sensitive to the actionof PMNs (because the PMNs are not killed when they get in contact withthe biofilm). We have previously shown that in vitro biofilms of QSdeficient bacteria can be phagocytosed by freshly isolated PMNs incontrast to QS proficient biofilms. Central for biofilm tolerance toPMNs is that rhamnolipid production forms a protective shield againstthe incoming PMNs and we have several data supporting that this is alsothe case. Using QS inhibitors should therefore greatly enhance theantimicrobial properties of the PMNs and allow them to efficientlyeradicate biofilm forming bacteria. Furthermore, rhamnolipid lyses thePMNs that subsequently spill out their content of DNA, hydrolyticenzymes and oxygen radicals. This creates an “evil circle” particularlywith respect to tissue damage, increasing inflammation and induction ofmutations in P. aeruginosa of which appearance of the mucoid phenotypesignificantly contributes to exacerbations.

The decrease in infection in the mouse experiments, the removal of invitro biofilm in a combinatorial experiment with tobramycin suggests thepotentials of using ajoene as a future antimicrobial drug for treatmentof chronic P. aeruginosa infections.

REFERENCES

-   W. C. Fuqua et al., J. Bacteriol., 1994, 176, p 269-275-   T. Bjarnsholt et al., Microbiology, 2005, 151, p. 3873-3880-   R. Naganawa et al., Applied and Environmental Microbiology, 62,    1996, p. 4238-4242-   EP 185324-   Block et al., J. Am. Chem. Soc., 1986, 108, p. 7045-7055-   U.S. Pat. No. 4,665,088-   WO 2010/100486-   M. Hentzer et al., EMBO J., 2003, 22, p. 3803-3815-   Rasmussen et al., Microbiology, 2005, 151, p. 1325-1340-   [V. E. Wagner et al., J. Bacteriol., 2003, 185, p. 2080-2095-   R. Rahim et al., Mol. Microbiol., 2001, 40, p. 708-718]-   B. B. Christensen et al., Methods Enzymol., 1999, 310, p. 20-42-   T. Brandt et al., Thorax, 1995, 50, p. 880-882-   P. M. Mendelman, 1985, Am Rev Respir Dis., 132, p. 761-765-   L. D. Christensen et al., Microbiology, 2007, 153, p. 2312-2320

1. A method of treating an infection of a biofilm forming bacteriacomprising providing a composition that comprises an (E,Z)-Ajoene offormula (1):

to a subject in need thereof. 2-20. (canceled)
 21. The method of claim1, wherein the bacteria is a gram negative bacteria.
 22. The method ofclaim 1, wherein the bacteria is selected from the group consisting ofVibrio fischeri, Aeromonas hydrophila, Aeromonas salmonicida,Agrobacterium tumefaciens, Burkholderia cepacia, Chromobacteriumviolaceum, Enterobacter agglomerans, Erwinia carotovora, Erwiniachrysanthemi, Erwinia Stewartii, Escherichia coli, Pseudomonasaureofaciens, Pseudomonas aeruginosa, Ralstonia solanacearum, Rhizobiumetli, Rhizobium leguminosarum, Rhodobacter sphaeroides, Salmonellatyphimurium, Serratia liquefaciens, Sinorhizobium meliloti, Vibrioanguillarum, Vibrio harveyi, Yersinia enterocolitica, and Yersiniapseudotuberculosis.
 23. The method of claim 1, wherein the bacteria isPseudomonas aeruginosa.
 24. The method of claim 1, wherein the bacterialinfection is a pulmonary infection.
 25. The method of claim 1, whereinthe bacterial infection is present in a chronic wound of said subject.26. The method of claim 1, wherein said subject has an immunedeficiency.
 27. The method according to claim 26, wherein the immunedeficiency is an immuno-compromising disease or said subject is takingan immuno-suppressant medication.
 28. The method according to claim 26,wherein the subject is selected from the group consisting of humans,pets and livestock.
 29. The method of claim 1, wherein the subject is amammal having cystic fibrosis.
 30. A composition comprising (E,Z)-ajoeneof formula (1):

and at least one antibiotic, wherein the antibiotic is selected from thegroup consisting of aminoglycosides, ansamycins, carbacephem,carbapenems, cephalosporins, glycopeptides, macrolides, monobactams,penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines.31. The composition according to claim 30, wherein the antibiotic is anaminoglycoside.
 32. The composition according to claim 30, wherein theantibiotic is selected from the group consisting of Ampicillin,Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin,Ticarcillin, Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam,Benzylpenicillin, Cloxacillin, Dicloxacillin, Methicillin, Oxacillin,Penicillin G, Penicillin V, Piperacillin Tazobactam, TicarcillinClavulanic Acid, Nafcillin, Cefadroxil, Cefazolin, Cephalexin,Cephalothin, Cephapirin, Cephradine, Cefaclor, Cefamandol, Cefonicid,Cefotetan, Cefoxitin, Cefprozil, Ceftmetazole, Cefuroxime, Loracarbef,Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoximeproxetil, Ceftazidime, Ceftizoxime, Ceftriaxone, Cefepime, Azithromycin,Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin,Troleandomycin, Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin,Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid,Gemifloxacin, Perfloxacin, Imipenem-Cilastatin, Meropenem, Aztreonam,Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin,Tobramycin, Paromomycin, Teicoplanin, Vancomycin, Demeclocycline,Doxycycline, Methacycline, Minocycline, Oxytetracycline, Tetracycline,Chlortetracycline, Mafenide, Silver Sulfadiazine, Sulfacetamide,Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole, Sulfamethizole, Rifampin, Rifabutin,Rifampin, Rifapentine, Linezolid, Quinopristin Dalfopristin, Bacitracin,Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazol,Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin,Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin,Ethionamide, Pyrazinamide, Para-aminosalicyclic acid, and Erythromycinethylsuccinate.
 33. The composition according to claim 30, wherein theantibiotic is tobramycin.
 34. A method of treating an infection of abiofilm forming bacteria comprising providing the composition of claim30 to a subject in need thereof.
 35. The method according to claim 34,wherein the bacteria is gram negative bacteria.
 36. The method of claim34, wherein the bacteria is selected from the group consisting of Vibriofischeri, Aeromonas hydrophila, Aeromonas salmonicida, Agrobacteriumtumefaciens, Burkholderia cepacia, Chromobacterium violaceum,Enterobacter agglomerans, Erwinia carotovora, Erwinia chrysanthemi,Erwinia Stewartii, Escherichia coli, Pseudomonas aureofaciens,Pseudomonas aeruginosa, Ralstonia solanacearum, Rhizobium etli,Rhizobium leguminosarum, Rhodobacter sphaeroides, Salmonellatyphimurium, Serratia liquefaciens, Sinorhizobium meliloti, Vibrioanguillarum, Vibrio harveyi, Yersinia enterocolitica, and Yersiniapseudotuberculosis.
 37. The method of claim 34, wherein the bacteria isPseudomonas aeruginosa.
 38. The method of claim 34, wherein thebacterial infection is a pulmonary infection.
 39. The method of claim34, wherein the bacterial infection is in a chronic wound of saidsubject.